6. PROJECT SUMMARY/ABSTRACT The 174-base bacteriophage 29 prohead RNA (pRNA) is an essential component of the motor that packages the 19-kilobase pair genomic DNA-gp3 complex (DNA-gp3) into the viral precursor capsid (prohead). This motor is one of the strongest molecular motors known, generating ~110 pN of force. pRNA forms a novel cyclic hexamer by intermolecular base pairing of identical molecules. This multimer binds to the head-tail connector of the prohead, where it appears as a pentameric ring by cryoEM-3D reconstruction. A multimer of the ATPase gp16 then binds to the pRNA oligomer to constitute the ATP-hydrolyzing subunits of the motor. pRNA is hypothesized to function in docking of the DNA-gp3 on the prohead, in recognition of the left end of DNA-gp3 to initiate packaging, and in assembly and enzyme function of the DNA translocating ATPase. pRNA exits the DNA-filled head during neck and tail assembly, and it is not a part of the mature virion. Study of the structure and function of this RNA-dependent DNA packaging motor may have general significance in uncovering targets for antiviral agents. The ultimate goal of the research is to determine the structural and functional roles of pRNA in the mechanism of DNA translocation. The current aims are: 1) X-ray crystallography will be used to determine atomic structures of monomeric and oligomeric pRNAs; 2) NMR will be used to determine the structure of the pRNA intermolecular pseudoknot and the essential CCA bulge of the A-helix; 3) Solution and site-directed hydroxyl radical probing will be used to map pRNA nucleotides in prohead/pRNA complexes with and without the ATPase gp16; 4) Site-directed and random pRNA mutants will be isolated to interrogate function of the pRNA in DNA translocation; and 5) Single-molecule laser tweezers studies of pRNA mutants having partial function will probe the roles of pRNA in 29 DNA translocation.